番茄CRISPR/Cas9介导的多基因编辑技术体系构建与应用

doi:10.16420/j.issn.0513-353x.2022-0339

摘要/Abstract

摘要：

为了构建番茄CRISPR/Cas9介导的多基因编辑体系，用SlU6-2p、SlU6-3p、SlU6-7p、SlU3-5p、SlU3-9p和SlU6-5p启动子分别替换pKSE401和pCBC-DT1T2载体中的拟南芥U6启动子，构建了1个双元载体（pMGET）、2个中间载体（pKC-S2M和pKC-S3M）和3个gRNA模块载体（pCBC-S1、pCBC-S2和pCBC-S3）。为了测试该多基因编辑体系，通过PCR扩增、Golden gate克隆和同尾酶技术，将含有6个番茄果实性状相关基因Green flesh（GF）、Ovate（O）、Locule number（LC）、SlMYB12（Y）、Tangerine（T）、Uniform ripening（U）靶点序列的sgRNA聚合构建多基因编辑载体pMGET-OYGTULC和pMGET-TULCOYG，检测6个基因同时被编辑的效率分别为44.00%和11.76%。用携带pMGET-OYGTULC载体的根癌农杆菌转化番茄材料获得2株6个基因均被编辑的植株，序列变异为单个碱基的插入、单个或多个碱基的缺失及大片段的缺失等多种形式。本研究中该多基因编辑体系可以高效地应用于番茄多基因编辑，并且可以得到稳定遗传的突变体，可为番茄基础研究和遗传改良提供一个简便的工具箱。

关键词: 番茄, CRISPR/Cas9, 多基因编辑, U3 snRNA基因, 同尾酶技术, GFP标记基因, 果实性状

Abstract:

In order to construct a CRISPR/Cas9 system for multiplex gene editing in tomato，Arabidopsis U6 promoters in pKSE401 and pCBC-DT1T2 vectors were replaced with SlU6-2p，SlU6-3p，SlU6-7p，SlU3-5p，SlU3-9p and SlU6-5p，respectively. Accordingly，a binary vector（pMGET），two intermediate vectors（pKC-S2M and pKC-S3M），and three gRNA module vectors（pCBC-S1，pCBC-S2 and pCBC-S3）were constructed. In order to test the system for multiplex gene editing in tomato，multiplex gene editing vectors pMGET-OYGTULC and pMGET-TULCOYG were constructed by aggregating sgRNA expression cassettes containing the target sequences of six tomato fruit trait-related genes Green flesh（GF），Ovate（O），Locule number（LC），SlMYB12（Y），Tangerine（T）and Uniform ripening（U），through PCR amplification，Golden gate cloning and isocaudamer technique，and the editing efficiency of six genes at the same time of the two vectors was 44.00% and 11.76%，respectively. The tomato material was transformed by Agrobacterium tumefaciens carrying pMGET-OYGTULC vector to obtain two plants with six genes edited. The sequence variations were in the form of single base insertion，single or multiple base deletion and large fragment deletion. In this study，the CRISPR/Cas9 system can be efficiently used for multiplex gene editing in tomato，and stable genetic mutants can be obtained，which can provide a versatile toolbox for basic research and genetic improvement in tomato.

Key words: tomato, CRISPR/Cas9, multiplex gene editing, U3 snRNA gene, isocaudamer technique, GFP marker gene, fruit trait

中图分类号:

S641.2

杨孟霞, 刘晓林, 曹雪, 魏凯, 宁宇, 杨沛, 李珊珊, 陈紫月, 王孝宣, 国艳梅, 杜永臣, 李君明, 刘磊, 李鑫, 黄泽军. 番茄CRISPR/Cas9介导的多基因编辑技术体系构建与应用[J]. 园艺学报, 2023, 50(6): 1215-1229.

YANG Mengxia, LIU Xiaolin, CAO Xue, WEI Kai, NING Yu, YANG Pei, LI Shanshan, CHEN Ziyue, WANG Xiaoxuan, GUO Yanmei, DU Yongchen, LI Junming, LIU Lei, LI Xin, HUANG Zejun. Construction and Application of a CRISPR/Cas9 System for Multiplex Gene Editing in Tomato[J]. Acta Horticulturae Sinica, 2023, 50(6): 1215-1229.

图/表 9

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引物Primer	引物序列（5′-3′）Primer sequence
MGET-17	TTCATCTCGGGTCTCGTTTGATCCACGATGATCTCCGTTGTTTTAGAGCTAGAAATAG
MGET-18	TTCATCTCGGGTCTCtAAACTCTGCCTCTCTTGATGCCCTGACCGTTGATAGTGGATAG
MGET-19	TTCATCTCGGGTCTCGCTTGTCCATTGCCACATTAGTGGGTTTTAGAGCTAGAAATAG
MGET-20	TTCATCTCGGGTCTCtAAACCCCAACTCAAGCCACAAAGTGACATATCCTACTGGTACG
MGET-21	TTCATCTCGGGTCTCGTTTGGTGAAGAACCTGCTATTCAGTTTTAGAGCTAGAAATAG
MGET-43	TTCATCTCGGGTCTCtAAACATTAGTAAACATCATGCCACAAACACACATGCTAATATTC
MGET-23	TTCATCTCGGGTCTCGTTTGCTTTGTGGCTTGAGTTGGGGTTTTAGAGCTAGAAATAG
MGET-24	TTCATCTCGGGTCTCtAAACTGAATAGCAGGTTCTTCACTGACCGTTGATAGTGGATAG
MGET-44	TTCATCTCGGGTCTCGCTTGTGGCATGATGTTTACTAATGTTTTAGAGCTAGAAATAG
MGET-26	TTCATCTCGGGTCTCtAAACAACGGAGATCATCGTGGATTGACATATCCTACTGGTACG
MGET-27	TTCATCTCGGGTCTCGTTTGGGGCATCAAGAGAGGCAGAGTTTTAGAGCTAGAAATAG
MGET-28	TTCATCTCGGGTCTCtAAACCCACTAATGTGGCAATGGACAAACACACATGCTAATATTC
ZP121	GAACAAAAGCCTTGAGAACC
ZP122	GTCGAAATCCCACACATCAC
ZP123	CAACTAACCTTATCACTGAATCC
ZP124	TGGTTAACTTTCCAGGGAGC
ZP125	GATGCTGGCATTTGTGTGATTC
ZP126	ACGACTAGAACCGTCAACGA
ZP129	CAAAGAGTGACATGCAATGC
ZP130	CCTCTTGAGAAGTAATGTTCC
ZP149	GCAGAGACAAAAAGAATGGG
ZP150	CATGATTAATCAAAGGAGGTTC
ZP191	GCCGAACACATCAACATTTC
ZP192	CCACACGTCTCGTCAGAATG
Cas9-909F	GCAGCTCTCCAAGGACACAT
Cas9-2450R	CGTGAGTTCTTCTGGCCCTT

引物Primer	引物序列（5′-3′）Primer sequence
MGET-17	TTCATCTCGGGTCTCGTTTGATCCACGATGATCTCCGTTGTTTTAGAGCTAGAAATAG
MGET-18	TTCATCTCGGGTCTCtAAACTCTGCCTCTCTTGATGCCCTGACCGTTGATAGTGGATAG
MGET-19	TTCATCTCGGGTCTCGCTTGTCCATTGCCACATTAGTGGGTTTTAGAGCTAGAAATAG
MGET-20	TTCATCTCGGGTCTCtAAACCCCAACTCAAGCCACAAAGTGACATATCCTACTGGTACG
MGET-21	TTCATCTCGGGTCTCGTTTGGTGAAGAACCTGCTATTCAGTTTTAGAGCTAGAAATAG
MGET-43	TTCATCTCGGGTCTCtAAACATTAGTAAACATCATGCCACAAACACACATGCTAATATTC
MGET-23	TTCATCTCGGGTCTCGTTTGCTTTGTGGCTTGAGTTGGGGTTTTAGAGCTAGAAATAG
MGET-24	TTCATCTCGGGTCTCtAAACTGAATAGCAGGTTCTTCACTGACCGTTGATAGTGGATAG
MGET-44	TTCATCTCGGGTCTCGCTTGTGGCATGATGTTTACTAATGTTTTAGAGCTAGAAATAG
MGET-26	TTCATCTCGGGTCTCtAAACAACGGAGATCATCGTGGATTGACATATCCTACTGGTACG
MGET-27	TTCATCTCGGGTCTCGTTTGGGGCATCAAGAGAGGCAGAGTTTTAGAGCTAGAAATAG
MGET-28	TTCATCTCGGGTCTCtAAACCCACTAATGTGGCAATGGACAAACACACATGCTAATATTC
ZP121	GAACAAAAGCCTTGAGAACC
ZP122	GTCGAAATCCCACACATCAC
ZP123	CAACTAACCTTATCACTGAATCC
ZP124	TGGTTAACTTTCCAGGGAGC
ZP125	GATGCTGGCATTTGTGTGATTC
ZP126	ACGACTAGAACCGTCAACGA
ZP129	CAAAGAGTGACATGCAATGC
ZP130	CCTCTTGAGAAGTAATGTTCC
ZP149	GCAGAGACAAAAAGAATGGG
ZP150	CATGATTAATCAAAGGAGGTTC
ZP191	GCCGAACACATCAACATTTC
ZP192	CCACACGTCTCGTCAGAATG
Cas9-909F	GCAGCTCTCCAAGGACACAT
Cas9-2450R	CGTGAGTTCTTCTGGCCCTT

基因名称 Gene name	基因ID Gene ID	靶点序列（5′-3′） Target sequence	PAM区 PAM area
Green flesh（GF）	Solyc08g080090	CCACTAATGTGGCAATGGA	CCT
Ovate（O）	Solyc02g085500	ATCCACGATGATCTCCGTT	GGG
Locule number（LC）	Solyc02g083950	ATTAGTAAACATCATGCCA	CCA
Tangerine（T）	Solyc10g081650	CCCAACTCAAGCCACAAAG	CCC
Uniform（U）	Solyc10g008160	GTGAAGAACCTGCTATTCA	TGG
SlMYB12（Y）	Solyc01g079620	GGGCATCAAGAGAGGCAGA	TGG

基因名称 Gene name	基因ID Gene ID	靶点序列（5′-3′） Target sequence	PAM区 PAM area
Green flesh（GF）	Solyc08g080090	CCACTAATGTGGCAATGGA	CCT
Ovate（O）	Solyc02g085500	ATCCACGATGATCTCCGTT	GGG
Locule number（LC）	Solyc02g083950	ATTAGTAAACATCATGCCA	CCA
Tangerine（T）	Solyc10g081650	CCCAACTCAAGCCACAAAG	CCC
Uniform（U）	Solyc10g008160	GTGAAGAACCTGCTATTCA	TGG
SlMYB12（Y）	Solyc01g079620	GGGCATCAAGAGAGGCAGA	TGG